DNA-mediated transfection techniques of NIH 3T3 cells have been used to demonstrate transforming genes in a number of human cancers. Most of these genes are cellular homologs of the viral ras gene which codes for the phosphoprotein p21. The yeast Saccharomyces cerevisiae contains two genes (RAS1 and RAS2) with significant homology to the mammalian ras gene. Yeast cells that lack their functional RAS genes fail to grow, indicating that RAS has some essential function. They will grow if they carry either the viral or mammalian ras genes. In addition, a modified RAS1 gene will induce transformation of mouse NIH 3T3 cells. This functional homology indicates that studies of the ras genes in yeast may be relevant to ras function in mammals. In this regard, our research aim is to identify genes whose products interact with the RAS proteins. Our specific aims are: (1) isolate conditional lethal mutation of the RAS genes either by in vivo or in vitro mutagenesis; (2) isolate revertants of the conditional RAS mutations. In particular, we will screen for cold-sensitive or warm-sensitive pseudorevertants of warm-sensitive or cold-sensitive RAS mutants, respectively. These pseudorevertants may identify genes whose gene products interact with the RAS protein. The phenotype of extragenic pseudorevertants will be determined independently from the RAS mutation. Pseudorevertants will be genetically mapped and cloned in order to identify their gene products; and (3) identify bypass mutants of RAS. We have isolated mutations in three genes which allow a yeast strain to grow without a functional RAS gene. The identification of the function of these genes may help identify the enzyme system(s) that RAS participates in. In this regard, the cloning and biochemical analysis of two genes reveal they are the adenylate cyclase structural gene and a gene affecting cAMP dependent protein kinase activity. We will continue our analysis of these two genes and a third gene in which mutations will allow yeast to survive without functional RAS. (X)